Herbicide degradation and copper complexation by bacterial mixed cultures from a vineyard stormwater basin
The use of stormwater basins as constructed wetlands for the bioremediation of agricultural runoff waters contaminated with pesticides has great potential. The structure and dynamics of the bacterial community in such system, and its function with respect to contaminant removal, remain to be investigated in detail.
Materials and methods
The bacterial component of sediment collected from a vineyard stormwater basin (in Rouffach, France) that pooled incoming runoff water containing pesticide and copper, was investigated by enumeration of cultivable bacteria on sediment extract solid medium and by liquid enrichment cultures from sediment, in the presence of glyphosate, diuron, 3,4-dichloroaniline and copper. Its structure, as a function of sediment location, depth, rhizospheric status and the presence of contaminants, was studied by temporal temperature gradient electrophoresis. Cultures obtained by enrichment were screened by RISA and RFLP and the ability of different cultures for contaminant mitigation was evaluated by the chrome azurol S method (copper complexation) and HPLC (glyphosate, diuron and 3,4-dichloroaniline degradation). The composition of the mixed cultures with the highest potential with regard to degradation of glyphosate, diuron and 3,4-dichloroaniline and copper complexation were evaluated by sequence analysis of cloned PCR-amplified 16S rRNA gene fragments obtained from enrichment cultures.
Results and discussion
The bacterial community structure of sediment showed differences depending on sampling location, sediment depth and sampling date. Spiking with a cocktail of concentrated glyphosate, diuron, 3,4-dichloroaniline and copper altered the bacterial community structure, but rhizospheric samples were less affected. RISA and RFLP analysis differentiated 98 distinct cultures, 28 of which were able to complex copper, and three, 35 and seven were able to degrade glyphosate, diuron and 3,4-dichloroaniline, respectively. Sequencing of cloned 16S rRNA gene fragments amplified from faster-growing rhizospheric mixed culture 106, selected as the most efficient in complexing copper and degrading glyphosate, diuron and 3,4-dichloroaniline, showed that it consisted of Arthrobacter sp., Pseudomonas putida, Delftia acidovorans and Brevundimonas sp. strains.
The investigated stormwater basin contains bacterial populations specifically adapted to the transformation of diuron, 3,4-dichloroaniline (3,4-DCA) and glyphosate, and to copper complexation. The mixed culture 106 complexed high amounts of copper ions and degraded glyphosate and diuron without accumulation of the major diuron metabolite 3,4-DCA. Our results also suggest that plants may help to stabilise bacterial-driven pesticide mitigation in environments subject to variable conditions such as stormwater basins.
KeywordsBioremediation Copper Diuron Glyphosate Ribosomal 16S rRNA gene Siderophores
- Aubertot JN, Barbier JM, Carpentier A, Gril JJ, Guichard L, Lucas P, Savary S, Savini I, Voltz, M (2005) Pesticides, agriculture et environnement. Réduire l’utilisation des pesticides et en limiter les impacts environnementaux. Rapport d’expertise scientifique collective. INRA et CEMAGREF, ParisGoogle Scholar
- Bazot S, Lebeau T (2008). Simultaneous mineralization of glyphosate and diuron by a consortium of three bacteria as free- and/or immobilized-cells formulations. Appl Microbiol Biotechnol 77:1351–1358Google Scholar
- Cardinale M, Brusetti L, Quatrini P, Borin S, Puglia AM, Rizzi A, Zanardini E, Sorlini C, Corselli C, Daffonchio D (2004) Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Appl Environ Microbiol 70:6147–6156CrossRefGoogle Scholar
- El-Fantroussi S, Verschuere L, Verstraete W, Top EM (1999) Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16SrRNA gene fingerprints and community-level physiological profiles. Appl Environ Microbiol 65:982–988Google Scholar
- Felske A, Akkermans ADL, De Vos WM (1998) Quantification of 16S rRNA in complex bacterial communities by multiple competitive reverse transcription-PCR in temperature gradient gel electrophoresis fingerprints. Appl Environ Microbiol 64:4581–4587Google Scholar
- Grégoire C, Elsaesser D, Huguenot D, Lange J, Lebeau T, Merli A, Mosé R, Passeport E, Payraudeau S, Schutz T, Schulz R, Tapia-Padilla G, Tournebize J, Trevisan M, Wanko A (2009) Mitigation of agricultural nonpoint-source pesticide pollution in artificial wetland ecosystems. Environ Chem Lett 7:205–231CrossRefGoogle Scholar
- Heuer H, Hartung K, Wieland G, Kramer I, Smalla K (1999) Polynucleotide probes that target a hypervariable region of 16S-RNA genes to identify bacterial isolates corresponding to bands of community fingerprints. Appl Environ Microbiol 65:1045–1049Google Scholar
- IFEN (2007) Les pesticides dans les eaux—données 2005. Les dossiers IFEN 9Google Scholar
- Kjelleberg S, Hermansson M (1984) Starvation-induced effects on bacterial surface characteristics. Appl Environ Microbiol 48:497–503Google Scholar
- Le Bissonnais Y, Gascuel-Odoux C (2000) L’érosion hydrique des sols cultivés en milieu tempéré. Sol : interface fragile, (Stengel P & Gelin S, eds.), pp. 222. Coédition INRA et Nathan, ParisGoogle Scholar
- Marugg JD, van Spanje M, Hoekstra WP, Schippers B, Weisbeek PJ (1985) Isolation and analysis of genes involved in siderophore biosynthesis in plant-growth-stimulating Pseudomonas putida WCS358. J Bacteriol 164:563–570Google Scholar
- McGrath SP, Shen ZG, Zhao FJ (1997) Heavy metal uptake and chemical changes in the rhizosphere of Thlaspi caerulescens and Thlaspi ochroleucum grown in contaminated soils. Plant Soil 188:153–159Google Scholar
- Obojska A, Lejczak B, Kubrak M (1999) Degradation of phosphonates by streptomycete isolates. Appl Microbiol Biotech 51:872–876.Google Scholar
- Schröder P, Navarro-Aviñó JHA, Goldhirsh AG, DiGregorio S, Komives T, Langergraber G, Lenz A, Maestri E, Memon AR, Ranalli A, Sebastiani L, Smrcek S, Vanek T, Vuilleumier S, Wissing F (2007) Using phytoremediation technologies to upgrade waste water treatment in Europe. Environ Sci Pollut Res 14:440–447CrossRefGoogle Scholar
- Stenström J, Svensson K, Johansson M (2001) Reversible transition between active and dormant microbial states in soil. FEMS Microbiol Ecol 36:93–104Google Scholar
- Tixier C, Sancelme M, Aït-Aïssa S, Widehem P, Bonnemoy F, Cuer A, Truffaut N, Veschambre H (2002) Biotransformation of phenylurea herbicides by a soil bacterial strain, Arthrobacter sp. N2: structure, ecotoxicity and fate of diuron metabolite with soil fungi. Chemosphere 46:519–526CrossRefGoogle Scholar
- Travkin VM, Golovleva LA (2003) The degradation of 3,4-dichloroaniline by Pseudomonas fluorescens strain 26-K. Microbiology 72:240–243Google Scholar
- USEPA (1996) Microwave assisted acid digestion of sediments, sludges, soils and oils, method 3051A. USEPA, Washington, DCGoogle Scholar
- Van Elsas JD, Torsvik V, Hartmann A, Øvreås L, Jansson JK (2007) The bacteria and archaea in soil. Modern soil microbiology, 2nd edn. CRC, United StatesGoogle Scholar
- Vauterin L, Vauterin P (1992) Computer-aided objective comparison of electrophoresis patterns for grouping and identification of microorganisms. Eur Microbiol 1:37–41Google Scholar